Treatment of murine SCC VII tumors with localized hyperthermia and temperature-sensitive liposomes containing cisplatin

The release of cisplatin (CDDP) encapsulated in temperature-sensitive unilamellar liposomes to murine SCC VII carcinoma by localized hyperthermia and the effects of the treatment on tumor growth were studied. A transition temperature of the temperature-sensitive liposomes containing cisplatin (LIP-CDDP) was 41 degrees C. Twenty-four hours after injection of LIP-CDDP, the heated tumors (42 degrees C, 60 min) contained 3.3 times more CDDP than the unheated tumors receiving free CDDP. Although the uptake of liposome-associated CDDP by liver was approximately threefold greater at 1.5 h after injection than uptake of free CDDP, it decreased about 50% over a 24-h period. No difference in uptake of the two forms of CDDP by kidney was observed. The combination of LIP-CDDP and localized heating at 42 or 43 degrees C was more effective relative to the amount of CDDP in delaying tumor growth than that of free CDDP and hyperthermia. Treatment with LIP-CDDP plus local heating resulted in a dose-modifying factor of 5.3 when compared with free CDDP and no hyperthermia. The dose-modifying factor was 2.8 when treatment with LIP-CDDP and heat was compared with treatment with free CDDP and heat. Thus CDDP could be released selectively from the temperature-sensitive liposomes by heat and resulted in both a greater uptake of the drug and a delay in tumor growth.     

Enhancement of cell killing by induction of apoptosis after treatment with mild hyperthermia at 42 degrees C and cisplatin.

Ohtsubo, T., Igawa, H., Saito, T., Matsumoto, H., Park, H. J., Song, C. W., Kano, E. and Saito, H. Enhancement of Cell Killing by Induction of Apoptosis after Treatment with Mild Hyperthermia at 42 degrees C and Cisplatin. Radiat. Res. 156, 103-109 (2001).We examined the interactive effects of cisplatin (1.0 microg/ml) combined with hyperthermia on cell killing and on the induction of apoptosis in IMC-3 human maxillary carcinoma cells. The cytotoxic effects of hyperthermia on IMC-3 cells at 44 degrees C were greater than at 42 degrees C, as has been reported for many other cells. The induction of apoptosis, DNA fragmentation and poly(ADP-ribose) polymerase cleavage were greater after hyperthermia at 44 degrees C for 30 min compared with treatment at 42 degrees C for 105 min, even though both of these heat doses were isoeffective in reducing cell survival to 50%. Treatment with cisplatin at 37 degrees C for up to 120 min did not result in cytotoxicity or the induction of apoptosis. The enhancement ratio for treatment with cisplatin at 42 degrees C was greater than that at 44 degrees C. More apoptosis was induced after the treatment with cisplatin at 42 degrees C compared to treatment with cisplatin at 44 degrees C. Taking these findings together, the combination of cisplatin and hyperthermia at 42 degrees C appeared to be more effective than cisplatin with hyperthermia at 44 degrees C for the induction of apoptosis in IMC-3 cells.     

Hyperthermia increases interleukin-6 in mouse skeletal muscle

Skeletal muscles produce and contribute to circulating levels of IL-6 during exercise. However, when core temperature is reduced, the response is attenuated. Therefore, we hypothesized that hyperthermia may be an important and independent stimulus for muscle IL-6. In cultured C2C12 myotubes, hyperthermia (42°C) increased IL-6 gene expression 14-fold after 1 h and 35-fold after 5 h of 37°C recovery; whereas exposure to 41°C resulted in a 2.6-fold elevation at 1 h. IL-6 protein was secreted and significantly elevated in the cell supernatant. Similar but reduced responses to heat were seen in C2C12 myoblasts. Isolated soleus muscles from mice, exposed ex vivo to 41°C for 1 h, yielded similar IL-6 gene responses (>3-fold) but without a significant effect on protein release. When whole animals were exposed to passive hyperthermia, such that core temperature increased to 42.4°C, IL-6 mRNA in soleus increased 5.4-fold compared with time matched controls. Interestingly, TNF-α gene expression was routinely suppressed at all levels of hyperthermia (40.5-42°C) in the isolated models, but TNF-α was elevated (4.2-fold) in the soleus taken from intact mice exposed, in vivo, to hyperthermia. Muscle HSP72 mRNA increased as a function of the level of hyperthermia, and IL-6 mRNA responses increased proportionally with HSP72. In cultured C2C12 myotubes, when heat shock factor was pharmacologically blocked with KNK437, both HSP72 and IL-6 mRNA elevations, induced by heat, were suppressed. These findings implicate skeletal muscle as a "heat stress sensor" at physiologically relevant hyperthermia, responding with a programmed cytokine expression pattern characterized by elevated IL-6.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.     

Thermosensitization by methylglyoxal bis(guanylhydrazone) in Chinese hamster cells

The modification of methylglyoxal bis(guanylhydrazone) (MGBG) by 42 degrees C hyperthermia-and/or radiation-induced cell killing was examined in Chinese hamster V-79 cells. At concentrations of more than 10 microM, cell survival decreased exponentially with increased MGBG exposure times. Cell lethality of MGBG (10 microM) was not specific for cell-cycle phases tested from G1/S through G2. When cells were treated with MGBG (10 microM) for 6 hr and then exposed to 42 degrees C hyperthermia with or without a 24-hr interval, cell survival decreased markedly compared with that for 42 degrees C alone. Cells became thermosensitive after MGBG treatment. Cells exposed to MGBG (10 microM) for 6 hr before or after X irradiation were slightly radiosensitive. When X irradiation was combined with MGBG and 42 degrees C hyperthermia, cells became more radiosensitive. From these results, it is suggested that MGBG may change the intracellular state to sensitize cells to the cytotoxic action(s) of hyperthermia.     

Hyperthermic enhancement of antibody-complement cytotoxicity against normal mouse B lymphocytes and its relation to capping

Normal mouse B lymphocytes were exposed to water-bath hyperthermia in vitro and examined for susceptibility to antibody-complement (Ab-C) cytotoxicity. Enhancement of Ab-C cytotoxicity was observed during heat treatment at 42 or 43 degrees C. Sensitivity to Ab-C cytotoxicity returned to normal levels by 2-3 hr post exposure to 42 degrees C. No such recovery was observed when cells were preheated at 43 degrees C for 40 min. The mechanism responsible for heat-induced enhancement of Ab-C cytotoxicity may be related to the way heat affects the redistribution of membrane-bound antigen-antibody (Ag-Ab) complexes. To investigate this possibility, cells were preheated at 37, 42, or 43 degrees C. The Ab-C assay was then performed at 37 degrees C immediately or 2.5 hr after hyperthermia. The distribution of Ag-Ab complexes was evaluated by immunofluorescence. A direct correlation was found between the hyperthermic enhancement of Ab-C cytotoxicity and the hyperthermic inhibition of capping, a process where membrane-bound Ag-Ab complexes coalesce into a polar cap on the cell surface. Sensitivity to Ab-C cytotoxicity returned to normal levels when cells restored the ability to cap Ag-Ab complexes following 42 degrees C hyperthermia. Cells heated at 43 degrees C were still sensitive to Ab-C cytotoxicity and did not recover the capping ability even 2.5 hr after heat treatment.     

Effects of m-aminobenzamide on the response of Chinese hamster cells to hyperthermia and/or radiation

The modifying effects of m-aminobenzamide (m-ABA), an inhibitor of poly(ADP-ribose) synthesis, on 42 degrees C hyperthermia- and/or radiation-induced cell killing were examined in Chinese hamster V-79 cells. When cells were exposed to 42 degrees C hyperthermia in combination with m-ABA (10 mM), cell survival decreased compared with that for 42 degrees C hyperthermia alone. Thermosensitizing effects of m-ABA changed with treatments in a decreasing order of during and after heating greater than during heating greater than after heating. Treatments with m-ABA during and/or after X irradiation enhanced radiation-induced cell killing. When cells were exposed to combined treatment with X irradiation, 42 degrees C hyperthermia (60 min), and m-ABA (24 hr), cell survival decreased markedly compared with that for X irradiation alone. However, with both X----42 degrees C and X----42 degrees C----m-ABA, the enhancement ratios (ER), designated as D0 ratio, were similar. These results suggest that the mechanisms of radiosensitization by m-ABA may be similar to those of 42 degrees C hyperthermia.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.     

Inhibitory effects of hyperthermia on mechanisms involved in autoresuscitation from hypoxic apnea in mice: a model for thermal stress causing SIDS.

The physiological mechanisms that might be involved in an association between heat stress and sudden infant death syndrome (SIDS) are obscure. We tested the hypothesis that a combination of acute hypoxia and elevated body temperature (T(B)) might prevent autoresuscitation from hypoxic apnea (AR). We exposed 21-day-old mice (total = 216) to hyperthermia (40.5-43.5 degrees C), hypoxia, or a combination of the two. Neither hyperthermia alone (40.5-42.5 degrees C) nor hypoxia alone was found to be lethal, but the combination produced failure to AR during the first hypoxic exposure with increasing frequency as T(B) increased. The ability to withstand multiple hypoxic exposures was also reduced as T(B) increased. In contrast, heat stress causing moderate T(B) increase (40.5 degrees C) had no effect on survival. Increased T(B) (43.5 degrees C) reduced gasping duration and number of gasps. It increased heart rate during anoxia but did not alter gasping rate. Furthermore, the oxygen-independent increase in heart rate observed before gasping failure was usually delayed until after the last gasp in hyperthermic animals. Mild dehydration occurred during T(B) elevation, but this did not appear to be a primary factor in AR failure. We conclude that a thermal stress, which by itself is nonlethal, frequently prevents AR from hypoxic apnea. This may be due, at least in part, to decreased gasp number and duration as well as to hyperthermia-related asynchrony of reflexes regulating heart and gasping frequencies during attempted AR.     

Hypoxic cell radiosensitization by moderate hyperthermia and glucose deprivation

Cell culture studies were carried out to determine whether moderate hyperthermia reduces the oxygen enhancement ratio of cells under well-defined cultural conditions. Using asynchronously growing HeLa cells, the OER of cells with and without glucose was determined following exposure of cells to moderate hyperthermia, 40.5 degrees C for 1 hr, immediately after X irradiation. The OER of cells with 5 mM glucose was 3.2, whereas the OER of glucose-deprived cells was reduced to 2.0. The pH of the cell culture medium was kept at 7.4 throughout the experiments. The present finding may provide a clue toward further enhancing the radiosensitization of hypoxic cells by heat.     

Enhanced expression of heat shock protein and mRNA synthesis by type I interferon in human HL-60 leukemic cells

The effects of IFN and mild hyperthermia on the responses of human promyelocytic HL-60 cells were investigated. Cells subjected to an elevated culture temperature (39.5 degrees-40.5 degrees C instead of 37 degrees C, herein referred to as heat-treated cells) showed an increase in heat shock proteins (HSPs) and corresponding mRNA synthesis, which were additionally potentiated by the presence of IFN. With cells cultured at 37 degrees C, IFN had no effect on HSP expression. The observed inhibition (40-70%) of RNA polymerase II-directed RNA synthesis (based on alpha-amanitin sensitivity) in isolated nuclei of heat-treated cells was also significantly reversed by the simultaneous addition of IFN. These data suggest that the IFN-amplified HSP gene expression may be involved in preventing irreversible damage or in fine tuning the recovery of mammalian cells from heat stress.     

Association of microwaves and ionizing radiation: potentiation of teratogenic effects in the rat

Pregnant Wistar rats were exposed to either microwave-induced hyperthermia or gamma radiation or a combination of both. In microwave-induced hyperthermia, a core temperature of 42 degrees C was slightly teratogenic and a core temperature greater than or equal to 43 degrees C was highly teratogenic. Gamma radiation at 40 cGy was subteratogenic, while a dose of 75 cGy was highly teratogenic. A combination of microwave-induced hyperthermia up to 42 degrees C with 40 cGy of gamma radiation was highly teratogenic, indicating a mutual potentiation of the embryotoxic action of these two teratogens.     

Local 42 degrees C hyperthermia improves vascular conductance of the R3230Ac rat mammary adenocarcinoma during sodium nitroprusside infusion

The effect of sodium nitroprusside-induced hypotension on the perfusion of the R3230 adenocarcinoma during local 42 degrees C hyperthermia was studied using a combination of intravital microscopy and laser Doppler flowmetry. Fischer 344 rats were implanted with dorsal skin flap window chambers containing the R3230Ac tumor and allocated to three treatment groups (34 degrees C with nitroprusside, 42 degrees C with nitroprusside, and 42 degrees C with 0.9% saline). After baseline observation at 34 degrees C, tumors were locally heated to 42 degrees C using a water bath and either 0.9% saline or nitroprusside sufficient to reduce blood pressure 20% below pretreatment baseline was infused. Nitroprusside at 34 degrees C decreased tumor vascular conductance 40% with no effect on the diameter of arterioles entering the tumor. The diameter of arterioles entering 42 degrees C heated tumors increased 35% independent of blood pressure change. Saline at 42 degrees C had no effect on tumor vascular conductance; however, nitroprusside at 42 degrees C increased tumor vascular conductance 55%. Local 42 degrees C tumor heating, combined with a moderate reduction in blood pressure with nitroprusside, overrides the vascular steal effect associated with reduced perfusion pressure alone and results in improved tumor perfusion. Observations of the effect of vasodilator substances on normothermic tumor perfusion cannot be extrapolated to situations where moderate hyperthermia is used.     

Sensitization to the cytotoxicity of adriamycin by verapamil and heat in multidrug-resistant Chinese hamster ovary cells.

Development of multidrug resistance to anticancer agents is a major limitation for the success of cancer chemotherapy. The chemosensitizer verapamil increases intracellular accumulation of drugs such as adriamycin in certain multidrug-resistant cell lines. When combined with verapamil, hyperthermia should be able to alter membrane permeability to adriamycin and to enhance the cytotoxicity of the drug. Verapamil increased the cytotoxicity of adriamycin in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) but not in drug-sensitive cells (AuxB1). Hyperthermia (42 degrees C) alone clearly increased the cytotoxicity of adriamycin in AuxB1 cells. There was also a small increase in CH(R)C5 cells at 42 and 43 degrees C. In drug-resistant cells, the cytotoxicity of adriamycin increased considerably when verapamil was combined with heat. This effect was dependent on temperature and increased with time of incubation. At 37 degrees C, verapamil increased the uptake of adriamycin in CH(R)C5 cells, while drug efflux decreased. When verapamil was combined with hyperthermia, drug efflux decreased even further. These results led to an overall increase in intracellular accumulation of the drug. In drug-sensitive cells, hyperthermia increased both the uptake and efflux of adriamycin, but verapamil had no effect. Verapamil plus heat increased the cytotoxicity of adriamycin in drug-resistant cells, and this was accompanied by altered permeability of the membrane to the drug. Hyperthermia combined with verapamil could be beneficial by increasing the effectiveness of adriamycin in the elimination of multidrug-resistant cells in a localized target region.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

Inhibitors of poly(ADP-ribose) synthetase enhance the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster cells

Two inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37 degrees C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5-10 min) 45 degrees C heat shock, and after 50 and 100 min exposures to 42 degrees C. When present during heating at 42 degrees C, the inhibitors reduced the shoulder of the 42 degrees C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.     

Modification of Cell Lethality at Elevated Temperatures The pH Effect

The lethal response of Chinese hamster ovary cells to hyperthermia was determined at selected extracellular pH. Decreasing pH from 7.6 to 6.7 increased the lethal response of cells over the temperature range of 41 to 44°C. Cell viability was not effected over this pH range at 37°C. The pH sensitizing affect was most prominent at temperatures which were marginally lethal at normal pH (7.4). Four hours of exposure to 42°C decreased survival to 10% at pH 7.4 and 0.01% at pH 6.7. Enhanced cell killing was observed when the cells were exposed to reduced pH and elevated temperatures simultaneously. Prolonging the time of pH exposure before and after hyperthermia did not influence survival. High-density culturing increased the sensitivity of cells to hyperthermia. This affect was due to metabolic acidification of the medium and could be reversed by adjusting the pH.     

Effect of step-down heating on brachytherapy in a murine tumor system

The effects of step-down heating combined with low-dose-rate irradiation (brachytherapy) were studied using a murine mammary adenocarcinoma (MTG-B) grown in the flanks of C3H mice. Treatment was initiated when tumors reached 0.9 to 1.1 cm in diameter. Step-down heating consisted of 7.5 min at 45 degrees C immediately followed by 7.5 min at 42 degrees C. Step-up heating consisted of 7.5 min at 42 degrees C immediately followed by 7.5 min at 45 degrees C. Step-down heating and step-up heating were compared to a single 45 degrees C, 15-min hyperthermia treatment. These hyperthermia protocols were combined before, in the middle of, or after brachytherapy. There were 4 untreated controls, 6 sham controls, and 11 treated animals in each of the brachytherapy-alone and combined treatment groups. The entire experiment was repeated at brachytherapy doses of 988, 1273, and 1603 cGy. In addition, the effects of step-down heating, step-up heating, and single-temperature hyperthermia were tested alone and in combination with sham treatment for each sequence. Based on daily measurements of tumor diameter, the growth delay to doubling volume was used as the biological end point. To compare the various treatment protocols, an isoeffect thermal enhancement ratio (TERiso) was calculated. Step-down heating after 988 cGy brachytherapy had a TERiso of 2.0 +/- 0.04, while step-up heating after 988 cGy brachytherapy had a TERiso of 1.7 +/- 0.05. Overall, the thermal enhancement ratios calculated from these growth delays indicate that step-down heating caused significantly greater hyperthermic radiosensitization than step-up heating when combined with brachytherapy.     

Thermal tolerance of contractile function in oxidative skeletal muscle: no protection by antioxidants and reduced tolerance with eicosanoid enzyme inhibition

Mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. This study identified the critical temperature, resulting in a loss of contractile function in isolated diaphragm (thermal tolerance), and then tested the hypotheses 1) that increased reactive oxygen species (ROS) production contributes to the loss of contractile function at this temperature, and 2) eicosanoid metabolism plays an important role in preservation of contractile function in hyperthermia. Contractile function and passive force were measured in rat diaphragm bundles during and after 30 min of exposure to 40, 41, 42 or 43 degrees C. Between 40 and 42 degrees C, there were no effects of hyperthermia, but at 43 degrees C, a significant loss of active force and an increase in passive force were observed. Inhibition of ROS with the antioxidants, Tiron or Trolox, did not inhibit the loss of contractile force at 43 degrees C. Furthermore, treatment with dithiothreitol, a thiol (-SH) reducing agent, did not reverse the effects of hyperthermia. A variety of global lipoxygenase (LOX) inhibitors further depressed force during 43 degrees C and caused a significant loss of thermal tolerance at 42 degrees C. Cyclooxygenase (COX) inhibitors also caused a loss of thermal tolerance at 42 degrees C. Blockage of phospholipase with phospholipase A(2) inhibitors, bromoenol lactone or arachidonyltrifluoromethyl ketone failed to significantly prevent the loss of force at 43 degrees C. Overall, these data suggest that ROS do not play an apparent role in the loss of contractile function during severe hyperthermia in diaphragm. However, functional LOX and COX enzyme activities appear to be necessary for maintaining normal force production in hyperthermia.     

The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.